Validation of the Sysmex XT‐2000iV hematology system for dogs,cats, and horses. II. Differential leukocyte counts

Background: The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that stains nucleic acids in leukocytes with a fluorescent dye. A 4‐part differential is obtained using side fluorescence light and laser side scatter. Objective: The purpose of this study was to validate the Sysmex XT‐2000iV for determining differential leukocyte counts in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV (Auto‐diff) and the CELL‐DYN 3500. Manual differentials were obtained by counting 100 leukocytes in Wright‐stained blood smears. Results: Leukocyte populations in the Sysmex DIFF scattergram were usually well separated in equine samples, but were not as well separated in canine and feline samples. Correlation among the Sysmex XT‐2000iV, CELL‐DYN 3500, and manual counts was excellent for neutrophil counts (r ≥.97) and good for lymphocyte counts (r ≥.87) for all three species. Systematic differences between the 3 methods were seen for lymphocyte and monocyte counts. The Sysmex reported incomplete differential counts on 18% of feline, 13% of canine, and 3% of equine samples, often when a marked left shift (>10% bands) and/or toxic neutrophils were present. Eosinophils were readily identified in cytograms from all 3 species. Neither the Sysmex nor the CELL‐DYN detected basophils in the 7 dogs and 5 cats with basophilia. Conclusions: The Sysmex XT‐2000iV automated differential leukocyte count performed well with most samples from diseased dogs, cats, and horses. Basophils were not detected. Immature neutrophils or prominent toxic changes often induced errors in samples from cats and dogs.     

Validation of the Sysmex XT‐2000iV hematology system for dogs,cats, and horses. I. Erythrocytes,platelets, and total leukocyte counts

Background: The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories. Objective: The purpose of this study was to validate the Sysmex XT‐2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV and the CELL‐DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts were done on an additional 73 feline and 55 canine samples, and compared with automated Sysmex results. Results: Hemoglobin concentration, RBC counts, and total WBC counts on the Sysmex were highly correlated with those from the CELL‐DYN (r≥0.98). Systematic differences occurred for MCV and HCT. MCHC was poorly correlated in all species (r=0.33–0.67). The Sysmex impedance platelet count in dogs was highly correlated with both the impedance count from the CELL‐DYN (r=0.99) and the optical platelet count from the Sysmex (r=0.98). The Sysmex optical platelet count included large platelets, such that in samples from cats, the results agreed better with manual platelet counts than with impedance platelet counts on the Sysmex. Canine reticulocyte counts on the Sysmex correlated well (r=0.90) with manual reticulocyte counts. Feline reticulocyte counts on the Sysmex correlated well with aggregate (r=0.86) but not punctate (r=0.50) reticulocyte counts. Conclusion: The Sysmex XT‐2000iV performed as well as the CELL‐DYN on blood samples from dogs, cats, and horses with a variety of hematologic abnormalities. In addition, the Sysmex detected large platelets and provided accurate reticulocyte counts.     

Clinical evaluation of the CA530-VET hematology analyzer for use in veterinary practice

BACKGROUND: The CA530-VET is a completely automated impedance cell hematology analyzer, which yields a 16-parameter blood count including a 3-part leukocyte differential. OBJECTIVES: The aim of this study was to examine the operational potential of the CA530-VET and its value for use in veterinary practice. METHODS: The analyzer was tested for blood carry-over, precision, and accuracy. Comparison methods included the CELL-DYN 3500, microhematocrit centrifugation, manual platelet (PLT) counting for feline and equine species, and a 100-cell manual WBC differential. Blood samples for comparison of the methods were obtained from 242 dogs, 166 cats, and 144 horses. RESULTS: The carry-over ratio (K) was 0.28% for RBC, 0.59% for PLT, 0.32% for WBC, and 0.18% for hemoglobin (HGB) concentration. Coefficients of variation (CVs) for within-batch precision and duplicate measurement of blood samples were clearly within the required limits, except for duplicate platelet counts in cats (8.7%) and horses (9.5%). The WBC count was in excellent agreement for dogs and horses and RBC count was in excellent agreement for horses. The accuracy of feline WBC counts was not acceptable, with the exception of values at the high end of the range. RBC counts in dogs and cats, and HGB concentration and MCV in all 3 species were sufficiently accurate. The CA530-VET HCT results were in excellent agreement with microhematocrit results in horses but exceeded the maximum allowed inaccuracy for cats and dogs. In all species, PLT counts established mechanically and manually were not in adequate agreement. Large differences were found between the CA530-VET and the manual differential percentage for lymphocytes and "mid-sized cells" (monocytes and basophilic granulocytes). CONCLUSIONS: The CA530-VET can be considered useful for routine canine, feline, and equine blood cell analyses. It should not be considered accurate, however, for PLT counts, feline total WBC counts in the subnormal and normal range, and leukocyte differentials, except for granulocytes.     

Differential leukocyte counts determined in chicken blood using the Cell-Dyn 3500

Background: Automated hematology instruments commonly are used for mammalian blood analysis, but there is a lack of accurate automated methods available for avian leukocyte analysis. Objective: The aim of this study was to validate differential leukocyte counts in blood from chickens using the Cell-Dyn 3500 hematology system and avian-specific software.
Methods: Blood samples were collected in lithium-heparin tubes from 2 groups (n = 84 and n = 139) of laying hens. Manual 200-cell differential counts were done on routinely-stained blood smears, and manual total granulocyte counts (heterophils and eosinophils) were done using an eosinophil stain in a counting chamber. Automated differential counts were done using VET 2.3, a research and development version of avian-specific software for the Cell-Dyn 3500. Results were analyzed using Pearson's correlation and difference plots.
Results: Automated granulocyte counts from the Cell-Dyn were in good agreement with manual granulocyte counts ( r = 0.93 and 0.80 for the 2 study groups). No correlation was found between automated and manual lymphocyte counts. Correlation coefficients for monocyte counts were 0.70 and 0.43. Conclusion: Automated leukocyte results from the Cell-Dyn using VET 2.3 software were not fully accurate. Total granulocyte counts may be of clinical usefulness, but results obtained for other parameters were unreliable.     

Evaluation of the ADVIA 120 for analysis of canine cerebrospinal fluid

BACKGROUND: Conventional techniques for canine cerebrospinal fluid (CSF) analysis require large sample volumes and are labor intensive and subject to operator variability. Objective: The purpose of this study was to evaluate the ADVIA120 CSF assay for analysis of canine CSF samples. METHODS: CSF samples collected from 36 healthy control dogs and 17 dogs with neurologic disease were processed in parallel using the automated assay and established manual methods using a hemocytometer and cytocentrifugation. Results for WBC (total nucleated cell) count, RBC count, and differential nucleated cell percentages were compared using Spearman rank correlation coefficients and Bland-Altman bias plots. RESULTS: Correlation coefficients for WBC and RBC counts were 0.57 and 0.83 for controls, and 0.92 and 0.94 for ill dogs, respectively. Coefficients for the percentages of neutrophils, lymphocytes, and monocytes were 0.53, 0.26, and 0.12 for controls and 0.77, 0.92, and 0.70 for dogs with neurologic disease. When data were combined (n=53), correlation coefficients were 0.86 and 0.91 for WBC and RBC counts, and 0.63, 0.43, and 0.30 for neutrophil, lymphocyte, and monocyte percentages. A 9.5% positive bias and 7.0% negative bias were obtained for the ADVIA 120 CSF assay for lymphocytes and macrophages in dogs with neurologic disease with Bland-Altman analysis. A 12.2% positive bias was found for lymphocyte percentage in dogs with neurologic disease. CONCLUSIONS: Manual and automated CSF assays had moderate to excellent correlation for WBC and RBC concentrations, but results were more variable for differential cell percentages. The ADVIA assay may be more useful for assessment of canine CSF with adjustment of cell differentiation algorithms.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

Spurious,marked leukocytosis in 2 cats with Heinz body hemolytic anemia

Two domestic shorthair cats were presented with anorexia and dehydration following ingestion of caramelized onions. Shared key findings from a CBC (ADVIA 2120), serum biochemistry, and urinalysis included a spurious, marked leukocytosis with discordant basophil (BASO) channel and peroxidase channel WBC counts, normal manual leukocyte counts, mild, non-regenerative anemia with discrepancies between automated and manual reticulocyte counts, an abundance of large Heinz bodies (HBs), and highly irregular scattergrams. Case 1 also demonstrated a markedly elevated mean corpuscular hemoglobin concentration (MCHC) and discrepancies between RBC hemoglobin indices. Spurious leukocyte results were confirmed through re-analysis of samples (including the acquisition of a new sample, use of an alternate analyzer (Sysmex XT-2000iV; Case 1 only), and evaluation of scattergrams and blood films (Cases 1 and 2). Repeatedly discrepant reticulocyte counts were also identified. In both cases, the erroneous BASO WBC counts, discrepancies in reticulocyte counts and RBC indices, and atypical scattergrams were interpreted to result from various effects of the HBs. These cases emphasize the importance of reviewing blood films, interpreting scattergrams, and the usefulness of duplicate methods for determining various measurands on hematology analyzers.     

Canine complete blood counts: a comparison of four in‐office instruments with the ADVIA 120 and manual differential counts

Background: With more use of bench‐top in‐office hematology analyzers, the accuracy of reported values is increasingly important. Instruments use varied methods for cell counting and differentiation, and blood smears may not always be examined. Objective: The purpose of this study was to compare canine CBC results using 4 bench‐top instruments (Hemavet 950, Heska CBC‐Diff, IDEXX LaserCyte, and IDEXX VetAutoread) with ADVIA 120 and manual leukocyte counts. Methods: EDTA‐anticoagulated canine blood samples (n=100) were analyzed on each instrument. Manual differentials were based on 100‐cell counts. Linear regression, difference plots, paired t‐tests, and estimation of diagnostic equivalence were used to analyze results. Results: Correlations of HCT, WBC, and platelet counts were very good to excellent between all in‐office instruments and the ADVIA 120, but results varied in accuracy (comparability). Hemavet 950 and Heska CBC‐Diff results compared best with ADVIA results and manual leukocyte differentials. HCT and platelet counts on the IDEXX VetAutoread compared well with those from the ADVIA. Except for neutrophil counts, leukocyte differentials from all instruments compared poorly with ADVIA and manual counts. Reticulocyte counts on the LaserCyte and VetAutoread compared poorly with those from the ADVIA. Conclusions: The Hemavet 950 and Heska CBC‐Diff performed best of the 4 analyzers we compared. HCT, WBC, and platelet counts on the LaserCyte had minimally sufficient comparability for diagnostic use. Except for neutrophils (granulocytes), leukocyte differential counts were unreliable on all in‐office analyzers. Instruments with a 5‐part leukocyte differential provided no added benefit over a 3‐part differential. Assessment of erythrocyte regeneration on the LaserCyte and VetAutoread was unreliable compared with the ADVIA 120.     

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels

Background: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers.
Objectives: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs.
Methods: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample.
Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 10⁹ platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum.
Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.     

Evaluation of equine hemograms using the ADVIA 120 as compared with an impedance counter and manual differential count

BACKGROUND: The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. OBJECTIVES: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. METHODS: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various breeds, both sexes, and 2-6 years of age. Samples were stored for up to 12 hours at 4 degrees C and then analyzed on the ADVIA 120 and the Hemat 8. A 100-cell to 200-cell differential leukocyte count was performed by 3 independent observers on May-Grünwald-Giemsa-stained smears. Intra-assay precision of the ADVIA 120 was determined by analyzing 5 replicates each of 10 of the blood samples. RESULTS: Results from the ADVIA were significantly higher than those from the impedance counter for RBC count, total WBC count, hemoglobin concentration, red cell distribution width, MCH, and MCHC, and significantly lower for HCT and platelet count. Significantly higher neutrophil and basophil counts and significantly lower lymphocyte counts were obtained with the ADVIA 120 compared with manual counts. Based on Passing-Bablok regression analysis, RBC and platelet counts were in good agreement between the 2 analyzers; a constant and proportional bias was present for other values. Coefficients of variation for erythrocyte parameters on the ADVIA were <1%, but were higher for platelet (6%), total WBC (2%), differential WBC (4%-30%), and reticulocyte (75%) counts. CONCLUSIONS: Results obtained with equine samples on the ADVIA 120 were comparable with those obtained on an impedance counter; reference intervals differed statistically but overlapped. The ADVIA had poor precision for reticulocyte and differential leukocyte counts such that the latter should always be verified on smears.     

Comparison between microscopic and automated differential leukocyte counts in the silver fox (Vulpes vulpes) and the blue fox (Alopex lagopus)

Differential leukocyte (WBC) counts in blood from clinically healthy silver foxes (n=32) and blue foxes (n=37) obtained from an automated hematology analyzer (Technicon H*1 Hematology System) with canine software were compared with microscopic differential WBC counts (M-diff). There was good agreement between the automated differential cell count (A-diff) and the M-diff for neutrophil and lymphocyte percentages. The correlation was lower for monocyte percentages and variable for eosinophil percentages. There was no significant difference between the A-diff and M-diff in either fox species. The A-diff counts were very precise, and may be a good alternative to the traditional M-diff for screening populations of clinically healthy foxes or for studies on stress and animal welfare. Intercept values, however, indicated a constant bias that must be taken into account before interpreting results based on different methods of analysis     

Comparison of manual and automated methods for determining platelet counts in dogs with macrothrombocytopenia.

Platelet counts were performed in 43 Cavalier King Charles Spaniels (CKCS, a breed predisposed to macrothrombocytopenia) and in 10 control dogs using 3 automated systems and 3 manual methods (erythrocyte-lysing agents + counting chamber or evaluation of blood smear). Good correlations were found between platelet counts using all methods (all P < 0.0001; R2 = 0.71-0.85). Best correlations were found between the manual methods. Significantly larger platelets were found in CKCS with platelet count < or = 100,000/microl when compared with control dogs and CKCS with platelet count > 100,000/microl (both P < 0.0001). All platelet counts--except when made with the 2 counting chamber methods--were underestimated at platelet counts < or = 100,000/microl.     

Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45

BACKGROUND: In dogs, flow cytometry is used in the phenotyping of immunologic cells and in the diagnosis of hemic neoplasia. However, the paucity of specific antibodies for myeloid cells and B lymphocytes and of labeled antibodies for multicolor techniques limits the ability to detect all leukocyte subpopulations. This is especially true for neoplastic and precursor cells. CD18 and CD45 are expressed on all leukocytes and are involved in cell activation, and together could be useful in helping determine cell lineage. OBJECTIVES: The purpose of this study was to double label canine blood for CD18 and CD45 and to use the differential expression of antigens to identify leukocyte populations in dogs with non-neoplastic and neoplastic hematologic diseases. METHODS: A template was developed using blood samples from 10 clinically healthy dogs and a back-gating technique. Differential leukocyte counts obtained with the template were compared with those obtained by manual and automated methods on blood samples from 17 additional healthy dogs. Blood samples obtained from 9 dogs with non-neoplastic (reactive) hematologic diseases and 27 dogs with hemic neoplasia were double stained for CD18 and CD45 using mouse anticanine CD18 monoclonal antibody (mAb) plus phycoerythrin-conjugated rat anticanine CD45 mAb and fluorescein isothiocyanate-conjugated rabbit antimouse IgG. Hemic neoplasms were diagnosed by cell morphology, and immunophenotypic and cytochemical markers. RESULTS: With the double label, neutrophils, eosinophils, monocytes, and T- and B-lymphocytes were identified. In reactive disorders, a population of activated neutrophils with high CD45 and CD18 expression was detected. In hemic neoplasia, cell lineage was easily determined, even in acute leukemia. CONCLUSIONS: Double labeling for CD18/CD45 may be useful as a screening method to evaluate hematologic diseases and help determine cell lineage, and to aid in the selection of a panel of antibodies that would be useful for further analysis.     

Modification and evaluation of a multichannel blood cell counting system for blood analysis in veterinary hematology

A multichannel, semiautomated, blood cell counting system (Coulter Counter Model S550) was modified for use in veterinary hematology by increasing both the erythrocyte and leukocyte aperture currents to 225 V and 195 V, respectively, followed by calibration with human blood. It was evaluated by use of 350 samples from dogs, cats, horses, and cows. Values for leukocyte count, erythrocyte count, mean corpuscular volume, and hematocrit generated by the S550 were compared with values generated by an automated multichannel counter with histogram capability and other reference procedures when appropriate. Mean differences for values between S550 and reference values were less than calibration tolerance limits for the instrument. Correlation coefficients were excellent for all values of each species. To assess behavior of leukocytes of the different species with respect to the counting threshold, leukocyte size distribution histograms were generated for all samples analyzed on the S550. Means for mean leukocyte volumes in diluent and lysing reagents were 55.5, 56.6, 67.4, and 72.8 fl for dogs, cats, horses, and cows, respectively. Canine leukocyte counts, because of small leukocyte size, were an average of 14% less for 5 samples analyzed on the unmodified instrument, compared with analysis after increasing the leukocyte aperture current. Leukocyte threshold failures attributable to interfering particles, resulting in falsely high counts, were recognized in 14%, 10%, 8% and 0% of feline, bovine, canine, and equine samples, respectively. The magnitude of error in these samples averaged 5% for cows and dogs, but was considered not important. However, leukocyte counts of feline samples in this group averaged 44% falsely high.     

Hematologic reference intervals for koi (Cyprinus carpio), including blood cell morphology, cytochemistry, and ultrastructure

BACKGROUND: Hematologic data are used routinely in the health care of humans and domestic mammals. Similar data for fish are largely fragmentary or have not been collected. OBJECTIVES: The primary purpose of this study was to determine hematologic reference intervals for koi, an ornamental strain of the common carp (Cyprinus carpio). Secondarily, the morphology, cytochemical reactions, and ultrastructure of koi blood cells were characterized. METHODS: A CBC was performed manually on heparin-anticoagulated blood specimens using Natt and Herrick's diluent and a Neubauer-ruled hemacytometer. Leukocyte differential counts were done on Wright-Leishman- and Diff-Quik-stained blood smears. Cytochemical reactions of koi leukocytes were determined using commercial kits. Transmission electron microscopy was performed to characterize the ultrastructural features of koi blood cells. RESULTS: Hematologic reference intervals were established for healthy koi for PCV (30-34%), hemoglobin concentration (6.3-7.6 g/dL), RBC count (1.7-1.9 X 10(6)/ microL), WBC count (19.8-28.1 X 10(3)/ microL), RBC indices, and differential leukocyte counts. Lymphocytes were the predominant leukocyte (accounting for up to 80% of all leukocytes), whereas eosinophils were rare. Basophils were positive with PAS staining. Naphthol AS-D chloroacetate esterase activity was observed only in eosinophils. alpha-Naphthyl butyrate esterase and beta-glucuronidase activities were positive in monocytes. Some lymphocytes were reactive for alpha-naphthyl butyrate esterase and acid phosphatase activity. Ultrastructurally, leukocytes, erythrocytes, and thrombocytes were identified on the basis of cytoplasmic organelles and granule appearance. CONCLUSION: Hematologic reference intervals and knowledge of the cytochemical reactions and ultrastructural characteristics of koi leukocytes will help standardize hematologic studies in this species.     

Automated differential leukocyte count in horses,cattle, and cats using the Technicon H-1E hematology system

The differential leukocyte counts performed by an automated hematology analyzer, the Technicon H-1E Hematology System, and traditional microscopic method (M-Diff) from blood samples of 129 horses, 40 cattle, and 140 cats were compared. The comparison was repeated after selected subsets of data were created by deleting samples with certain patterns suggesting error with the automated differential cell count (A-Diff). The two methods had good comparison of results for neutrophils and lymphocytes in all three species. Results for equine monocytes correlated moderately well between the two methods and the correlation improved in the selected data set Monocyte results did not compare well for the bovine and feline samples. The A-Diff for feline eosinophils was inaccurate. The A-Diff may be accurate for bovine and equine eosinophils but too few examples of eosinophilia were present in the sample set to prove this. Basophils were too rarely seen in cattle and horses to validate A-Diff accuracy, but basophilia identified by the M-Diff in a cat was not identified by the A-Diff.     

Differential white blood cell counts in rabbits: a comparison of the Advia 2120 and a manual method

We evaluated the performance of the Advia 2120 (Siemens) differential leukocyte count (A-Diff) compared to the manual method (M-Diff) in rabbits. EDTA-anticoagulated blood samples collected for diagnostic purposes were analyzed within 6 h of collection. The M-Diff was performed blindly by 2 observers on blood smears by counting 200 cells. We initially included 117 samples; 25 samples were excluded because of suboptimal gating of leukocytes in the Advia peroxidase cytogram or poor blood smear quality. The correlation between the A-Diff and M-Diff was very high for heterophils (r = 0.924, p < 0.001) and lymphocytes (r = 0.903, p < 0.001), high for basophils (r = 0.823, p < 0.001), moderate for monocytes (r = 0.645, p < 0.001), and low for eosinophils (r = 0.336, p = 0.001). The Passing–Bablok regression analyses revealed a small-to-moderate constant error for lymphocytes and a slight constant error for basophils. Small proportional errors were detected for heterophils, lymphocytes, and eosinophils. The Bland–Altman analyses revealed that the Advia significantly underestimates heterophils and overestimates lymphocytes compared to M-Diff. The biases for the other leukocytes were minimal and likely clinical insignificant; however, our results, particularly for eosinophils, should be interpreted cautiously given the observed low percentages in our samples. Given the observed biases in heterophil and lymphocyte percentages in the Advia 2120 CBC results in rabbits, method-specific reference intervals should be used. The Advia can recognize leporine basophils. Evaluation of blood smears is still recommended to investigate abnormal results and erroneous cytograms reported by the Advia.     

Evaluation of methods of platelet counting in the cat

Platelet counts were performed in 50 cats presented for diagnostic investigation. For each cat, counts were obtained using a manual haemocytometer method and compared with counts obtained by estimation from a stained blood smear, a QBC VetAutoread analyser, a Zynocyte VS/2000 analyser, impedance automated counts on a Baker System using both EDTA and citrated anticoagulated blood, and use of a Zynostain modified counting chamber kit. None of the methods gave high correlation with the haemocytometer counts. The blood smear estimation of platelet counts had the highest correlation (r = 0.776) and was the only method to have reasonable values for both sensitivity and specificity. With the impedance automated counts, citrated anticoagulated blood had marginally higher correlation than EDTA anticoagulated blood, and the time between blood sampling and platelet count determination had no effect on the count obtained. When in-house analyser or impedance automated platelet counts are abnormal or not consistent with clinical findings, the authors recommend that a manual platelet count using either haemocytometry or examination of a blood smear is performed.     

Spurious reticulocyte profiles in a dog with babesiosis

A 9‐year‐old, female Maltese dog was referred to the Veterinary School of Toulouse with a 2‐day history of anorexia and weakness. On clinical examination, the dog had hyperthermia (39.7°C), abdominal discomfort, and polypnea. Significant laboratory findings included pigmenturia, hyperbilirubinemia, hypercreatininemia, hyperfibrinogenemia, abnormal Snap canine pancreas‐specific lipase, and pancytopenia with a nonregenerative anemia. A peripheral blood smear revealed numerous intraerythrocytic large Babesia but no polychromasia. There was a discrepancy between the absolute automated reticulocyte count (Sysmex reticulocyte count: 60 × 10⁹/L; RI 19.4–150.1 × 10⁹/L) and the manual reticulocyte count (3.6 × 10⁹/L) as well as the absence of polychromasia. The optical red blood cell scattergram showed an abnormal isolated reticulocyte cluster at the location of low‐fluorescence ratio cells. These findings were interpreted as erythrocytes parasitized by large Babesia. The discrepancy between the Sysmex reticulocyte count and the manual reticulocyte count has been reported previously in people with falciparum malaria and numerous intra‐erythrocytic Plasmodium falciparum organisms. This spurious reticulocyte profile and reticulocyte count were observed with the Sysmex XT‐2000iV and the ProCyte using the same fluorescent dye polymethine but not with the LaserCyte using new methylene blue which does not stain Babesia organisms on a blood smear performed for manual reticulocyte counting.     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.